Membrane fusion reactions are common in eukaryotic cells. Membranes are fused intracellularly in processes including endocytosis, organelle formation, inter-organelle traffic, and constitutive and regulated exocytosis. Intercellularly, membrane fusion occurs during sperm-egg fusion and myoblast fusion.
Membrane fusion has been induced artificially by the use of liposomes, in which the cell membrane is fused with the liposomal membrane, and by various chemicals or lipids, which induce cell-cell fusion to produce heterokaryons. Naturally occurring proteins shown to induce fusion of biological membranes are mainly fusion proteins of enveloped viruses.
In liposome-based delivery systems, liposomes are used to encapsulate bioactive molecules inside lipid vesicles for delivery into the cell. There has been interest in using such delivery systems in treating various cancer, since, in theory, the use of liposomes will allow for a more targeted approach to treating the cancer cells (Arias et al., Curr Drug Targets, Mar. 28 (2011)). However, the polar lipid head groups oriented on both surfaces of the lipid bilayer, along with an associated water layer, make spontaneous membrane fusion thermodynamically unfavorable. Moreover, the generalized release of the encapsulated bioactive molecules to both the cancerous cells and the healthy cells in a cancer inflicted tissue makes traditional liposome-based delivery systems less than perfect.
Various chemicals or lipids have been used to promote membrane fusion. However, these reagents usually exhibit cytotoxic effects (see, for example, Iwanoto et al., in Biol. Pharm. Bull. 19:860-863 (1996) and Mizugucji et al., in Biochem. Biophys. Res. Commun., 218:402-407 (1996)).
It is generally believed that membrane fusion under physiological conditions is protein-mediated. This has lead to the development of liposomes that contain fusion-promoting proteins (proteoliposomes), with decreased cytotoxicity (see, for example, Cheng, Hum. Gene Ther. 7:275-282 (1996); Hara et al., Gene 159:167-174 (1995); and Findeis et al., Trends Biotechnol., 11:202-205 (1993)).
One particularly interesting group of proteins recently identified as fusion-promoting proteins are the fusion-associated small transmembrane (FAST) proteins. The FAST proteins are a unique family of membrane fusion proteins encoded by the fusogenic retroviruses (Duncan et al., Virology 319:131-140 (2004). Currently, the FAST proteins include: p10, p14, p15 and p22. At 95 to 198 amino acids in size, the FAST proteins are the smallest known viral membrane fusion proteins. Rather than mediating virus-cell fusion, the FAST proteins are non-structural viral proteins that are expressed on the surfaces of virus-infected or -transfected cells, where they induce cell-cell fusion and the formation of multinucleated syncytia. A purified FAST protein, when reconstituted into liposome membranes, induces liposome-cell and liposome-liposome fusion, indicating the FAST proteins are bona fide membrane fusion proteins (Top et al., EMBO J. 24:2980-2988, 2005).
In contrast to most enveloped viral fusion proteins in which the cytoplasmic tail is extremely short relative to the overall size of the protein, the FAST proteins all have an unusual topology that partitions the majority of the protein to the membrane and cytoplasm, exposing ectodomains of just 20 to 43 residues to the extracellular milieu (Corcoran and Duncan, J. Virol 78(8):4342-51, 2004; Dawe et al., J Virol 79(10): 6216-26, 2005). Despite the diminutive size of their ectodomains, both p14 and p10 encode patches of hydrophobicity (HP) hypothesized to induce lipid mixing analogously to the fusion peptides encoded by enveloped viral fusion proteins (Corcoran et al., J Biol Chem 279(49): 51386-94, 2004; Shmulevitz et al., J Virol 78(6):2808-18, 2004). The p14 HP is comprised of the N-terminal 21 residues of the protein, but peptides corresponding to this sequence require the inclusion of the N-terminal myristate moiety to mediate lipid mixing. Nuclear magnetic resonance (NMR) spectroscopy revealed that two proline residues within the p14 HP form a protruding loop structure presenting valine and phenylalanine residues at the apex and connected to the rest of the protein by a flexible linker region (Corcoran et al., J Biol Chem 279(49): 51386-94, 2004). The p10 HP on the other hand, flanked by two cysteine residues that form an intramolecular disulfide bond, may have more in common with the internal fusion peptides of the Ebola virus and avian leukosis and sarcoma virus (ALSV) glycoproteins (Delos et al., J Virol 74(4): 1686-93, 2000; Delos and White, J Virol 74(20):9738-41, 2000; Gallaher, 1996; Ito et al., J Virol 73(10):8907-12, 1999; Ruiz-Arguello et al., J Virol 72(3): 1775-81, 1998), and likely adopts a cystine-noose structure that forces solvent exposure of conserved valine and phenylalanine residues for membrane interactions (Barry et al., J Biol Chem 285:16424, 2010). In contrast to p14 and p10, the 20 residue ectodomain of p15 completely lacks a hydrophobic sequence that could function as a traditional fusion peptide (Dawe et al., J Virol 79(10): 6216-26, 2005). In the absence of such a motif, the p15 ectodomain instead encodes a polyproline helix that has been proposed to function as a membrane destabilizing motif.
There is a need in the art for the targeted delivery of bioactive molecules encapsulated by liposomes.